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JFS C: Food Chemistry
Physicochemical Properties and Antioxidant
Capacity of 3 Polysaccharides fromGreen Tea,
Oolong Tea, and Black Tea
HAIXIA CHEN, ZHISHUANG QU, LINGLING FU, PENG DONG, AND XIN ZHANG
ABSTRACT: Three polysaccharide-rich fractions named GTPS, OTPS, and BTPS were isolated from green tea, oo-
long tea, and black tea, respectively. Chemical characteristics, glycosidase inhibitory effects, and antioxidant prop-
erties of the 3 fractions were compared. Monosaccharides of GTPS were composed of D-rhamnose, L-arabinose,
D-xylose, D-mannose, D-galactose, and D-glucose. But there were no xylose and mannose detected in OTPS and
BTPS. The molecular weight distributions were decreased from 9.2 to 251.5 KDa to 3.8 to 32.7 KDa with the fer-
mentation of the tea fromgreen tea to black tea. BTPS showed the highest -glucosidase inhibitory activity, antiox-
idant activities on hydroxyl radicals and DPPH radicals. The differences in antioxidant activities and glycosidase
inhibitory properties among the 3 polysaccharide-rich fractions appeared to be related to differences in monosac-
charide composition and molecular weight distribution of the polysaccharide.
PRACTICAL APPLICATION: Diabetes mellitus (DM) is one of the primary threats to human health due to its increas-
ing prevalence, chronic course, and disabling complications. Control of postprandial hyperglycemia and inhibition
of oxidative stress are suggested to be important in the treatment of diabetes. Many efforts had been made to search
for effective and safe -glucosidase inhibitors and antioxidants from natural materials to develop a physiological
functional food or lead compounds for curing diabetes. Coarse tea was used to cure diabetics in people in China and
Japan. The hypoglycemic activity increased with the contents of polysaccharide in coarse tea. Many studies have
focused on the hypoglycemic activities of tea polysaccharides, but little is known about the glycosidase inhibitory
effects of tea polysaccharide. The aim of this study was to find a tea polysaccharide with the best potential for ex-
ploitation in curing diabetes.
Keywords: antioxidant activities, composition, glycosidase inhibitory effects, tea polysaccharide
Introduction
D
iabetes mellitus (DM) is one of the primary threats to human
health due to its increasing prevalence, chronic course, and
disabling complications. Control of postprandial hyperglycemia
and inhibition of oxidative stress are suggested to be important in
the treatment of diabetes. Many efforts hadbeenmade to searchfor
effective and safe -glucosidase inhibitors and antioxidants from
natural materials to develop a physiological functional food or lead
compounds for curing diabetes (Hays and others 2008; Kwon and
others 2008).
Tea (Camellia sinensis L.) is the 2nd most consumed bever-
age in the world next to water. The tea plant has been widely
used for centuries by ancient cultures for its medicinal properties.
Tea is popularly consumed in unfermented (green tea), semifer-
mented (oolong tea), and fermented (black and pu-erh or red)
forms (Zhu and others 2002). The chemical composition of tea in-
cludes proteins, polysaccharides, polyphenols (catechins or flavan-
3-ols, theaflavins, thearubigins, and proanthocyanidins), chloro-
phyll, minerals and trace elements, volatile compounds, amino
and organic acids, lignins, and alkaloids (caffeine, theophylline,
and theobromine) (Seeram and others 2006). Tea was found to
MS 20090126 Submitted 2/12/2009, Accepted 5/4/2009. Authors are with
Tianjin Key Laboratory for Modern Drug Delivery & High-Efficiency,
School of Pharmaceutical Science and Technology, Tianjin Univ., Tian-
jin, 300072, P.R. China. Direct inquiries to author Chen (E-mail:
chennhxx@yahoo.com.cn).
have bioactivities including antioxidant (Yen and others 1997),
improving immune response (Bhattaxharyya and others 2004),
anti-atherosclerosis (Curin and Andriantsitohaina 2005), antihy-
pertension (Hodgson and others 2005), anti-infectious diseases
(Weber and others 2003), and antidiabetic properties (Anderson
and Polansky 2002) in recent studies. The chemical compositions
of the tea in different forms were different and induced the change
of the bioactivities (Yanagimoto and others 2003).
The control of postprandial hyperglycemia is critical in the early
therapy for diabetes. One therapeutic approach to decrease post-
prandial hyperglycemia is to retard absorption of glucose through
inhibition of carbohydrate hydrolyzing enzymes, for example, -
amylase and -glucosidase, in the digestive organs (Saito and oth-
ers 1998). In addition, several -glucosidases have been recently
screened and developed from natural sources (Lee and Lee 2001;
Kim and others 2004). Coarse tea was used to cure diabetics in
people in China and Japan. The hypoglycemic activity increased
with the contents of polysaccharide in coarse tea. Many studies
have been focused on the hypoglycemic activities of tea polysac-
charides (Chen and others 2005; Zhou and others 2007). But little
was known about the glycosidase inhibitory effects of tea polysac-
charide. In our previous studies, the chemical properties and the
antioxidant, hypoglycemic activities of the polysaccharide from
green tea were investigated (Chen and others 2004; Chen and oth-
ers 2008). But there was no report on the comparative study of the
composition and the bioactivities of the polysaccharides from dif-
ferent forms of tea. The objective of this study was to isolate the
C
2009 Institute of Food Technologists
R
Vol. 74, Nr. 6, 2009JOURNAL OF FOOD SCIENCE C469
doi: 10.1111/j.1750-3841.2009.01231.x
Further reproduction without permission is prohibited
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Physicochemical properties and antioxidant capacity. . .
polysaccharide-rich fraction from green tea, oolong tea, and black
tea, characterize the chemical composition, and evaluate the gly-
cosidase inhibitory effects and antioxidant activities of the 3 kinds
of tea polysaccharides (TPS).
Materials and Methods
Materials and chemicals
Green tea, oolong tea, and black tea were obtained from the
local tea market (They were produced by the Huangshan Moun-
tain Tea Factory, Anhui, China). -Glucosidase fromSaccharomyces
cerevisiae, p-Nitrophenyl -D-glucopyranoside as a synthetic sub-
strate of -glucosidase and pepsin were purchased from Sigma
Chemical Co. (St. Louis, Mo., U.S.A.). Trifuoroacetic acid (TFA)
1, 1- Diphenyl-2-picrylhydrazyl (DPPH), 2-deoxy-ribose, the stan-
dard monosaccharides (D-rhamnose, L-arabinose, D-galactose, D-
xylose, D-mannose, and D-glucose) and T-series Dextran standards
were obtained from China Sigma-Aldrich (Shanghai, China). All
other chemicals and reagents were purchased locally and were of
analytical grade.
Extraction and isolation of tea polysaccharide
Polysaccharides from green tea (GTPS), oolong tea (OTPS), and
black tea (BTPS) were obtained in the same procedure. Briefly, dry
meshed tea powders (100 g) were placed in a 3-L round-bottom
flask with 1.0 L of ethanol (80%, v/v) for 24 h to remove most of
the polyphenols and monosaccharide. After the supernatant was
removed, the residues were dried in air and then extracted with
hot water at 70
C for 60 min 3 times. The aqueous extracts were
concentrated and then precipitated with 4-fold volumes of 95%
ethanol. The precipitate that formed was collected by centrifuga-
tion at 3000 g for 10 min and repeatedly washed sequentially with
ethanol, acetone, and ether, respectively. The precipitate was dis-
solvedinhot water, andexcludedproteinwithSevag method(Staob
1965), and dialyzed against distilled water for 48 h with dialysis tub-
ing (molecular weight cut-off, 8000 Da) to remove low-molecular
weight matters, and then concentrated and precipitated with 4-
fold volumes of 95%ethanol to obtain the polysaccharide-enriched
fraction. The yield of tea polysaccharides from green tea, oolong
tea, and black tea were about 4.0, 4.6, and 4.2 g, respectively.
Composition analysis
Total sugar content was determined by the phenol-sulfuric
acid analysis using D-glucose as standard (Dubois and others
1956). Uronic acid content was determined by the carbazole-
sulfuric acid method using galacturonic acid as standard (Bitter
and Muir 1962). Protein was analyzed by the method of Brad-
ford (1976) using bovine serum albumin as the standard. Assay
of polyphenolic compounds in tea polysaccharide was conducted
according to Bonvehis method (Bonvehi and Coll 1997). The
composition of neutral monosaccharide was measured by gas
chromatography after converting them into acetylated derivatives
(Chaplin and Kennedy 1994). Briefly, 10 mg of polysaccharide were
hydrolyzed in a sealed glass tube with 2 Mtrifluoroacetic acid (TFA)
at 105
C for 10 h. The hydrolysate was evaporated to dryness.
The acid was removed under reduced pressure by repeated co-
evaporations withmethanol. The hydrolysates were thenconverted
into alditol acetates according to conventional procedures. Gas
chromatography was performed on a Shimadzu GC-14B instru-
ment with OV-1701capillary column (30 m 0.32 mm 0.5 m).
The temperature program was set to increase from 150 to 240
C at
an increment of 5
C/min and N
2
was the carrier gas. The standard
monosaccharides were used as references and they were measured
following the same procedure as the sample.
Determination of the molecular weight of tea
polysaccharide
For molecular weight determination of tea polysaccharides, high
performance gel permeation chromatography was performed on a
Shimadzu LC-20A chromatograph equipped with a Shodex OH pak
SB-804 column(7.8 300 mm; Showa Denko, Kawasaki, Japan) and
an RI detector (Polymer Lab. Ltd., Tokyo, Japan). Twenty microliters
of the polysaccharide were injected and eluted at a buffer (0.2 M
NaAc-AcOH, pH 6.0) flow rate of 0.5 mL/min at 35
C. The HPGPC
system was precalibrated with T-series Dextran standards (T-10, T-
40, T-70, T-110, and T-500).
Tea polysaccharide imaging with atomic
force microscopy
Tea polysaccharide was imaged with atomic force microscopy
according to the method of Zhang and others (2007). A stock so-
lution (1 mg/mL) was prepared by adding tea polysaccharide into
double distilled water. The solution was diluted to the final con-
centration of 1.0 g/mL. About 5 L of diluted tea polysaccharide
solution were dropped on the surface of mica sample carrier, al-
lowed to dry, and then imaged in air at room temperature. The
atomic force microscopy used in this study was an Ambios D3100
instrument (Ambios, Santa Cruz, Calif., U.S.A.) and was operated in
the tapping-mode. The resulting imaging force was estimated to be
0.05 to 3 nN and the resonant frequency was about 2 KHz.
Uv-vis and IR-FTR spectrophotometric analysis
The Uv-vis spectrophotometric analysis was conducted accord-
ing to the method of Yang and others (2008). Each tea polysac-
charide sample (1.0 mg) was dissolved in 10 mL of distilled water.
The absorbance of each sample solution was read over the range
of 190 to 900 nm, using a uv-2450 PC UV-visible spectrophotome-
ter (Shimadzu, Tokyo, Japan). FT-IR spectra (in KBr pellets, 2 mg
sample/200 mg KBr) were measuredusing the Bio-RadMerlinspec-
trophotometer operating at 4 cm
1
resolution.
Protease treatment of 3 polysaccharide fractions
Fifty micrograms of TPS were dissolved in PBS buffer (pH = 6)
and treated with 3 mg of pepsin (800-2500 U/mg protein, Sigma) at
55
C for 240 min. The reaction was stopped by increasing the tem-
perature to 90
C for 60 min to make protease inactive. After dial-
ysis with distilled water for 24 h, the polysaccharide solution was
concentrated, precipitated with 95% of ethanol, and freeze-dried.
The enzyme hydrolyzed fractions of TPS were obtained and tested
the scavenging effects on DPPH radicals at the concentration of 1
mg/mL. The same amount of pepsin treated with the same proce-
dure was used as the control.
Inhibition assay for -glucosidase activity
Inhibition assay of tea polysaccharide on -glucosidase activity
was performed according to Kim and others (2004). -Glucosidase
(0.25 unit) was premixed with tea polysaccharides at various con-
centrations, and 2 mM p-nitrophenyl -D-glucopyranoside as a
substrate in phosphate buffer was added to the mixture to start
the reaction. The reaction was incubated at 37
C for 30 min and
stopped by adding 2 mL of 0.1 M Na
2
CO
3
. -Glucosidase activ-
ity was determined by measuring release of p-nitrophenol from p-
nitrophenyl -D-glucopyranoside at 400 nm. Acarbose was used as
positive control.
C470 JOURNAL OF FOOD SCIENCEVol. 74, Nr. 6, 2009
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Physicochemical properties and antioxidant capacity. . .
Scavenging effects on hydroxyl radicals
Scavenging effects of tea polysaccharide on hydroxyl radicals
were performed as described by Halliwell and others (1987). Re-
action mixtures in a final volume of 1 mL contained deoxyribose
(60 mM), KH
2
PO
4
-KOH buffer (pH 7.4, 20 mM), FeCl
3
(100 M),
EDTA (100 M), various concentrations of tea polysaccharide sam-
ples, H
2
O
2
(1 mM), and ascorbic acid (100 M). After incubation at
37
C for 1 h, the color was developed by adding 1 mL of 1% thio-
barbituric acid (TBA) (w/v) and 1 mL of 25% (v/v) HCl, which was
then heated in a boiling water bath for 15 min. The absorbance of
resulting solution was measured spectrophotometrically at 532 nm
and butylated hydroxyl anisole (BHA) was used as positive control.
Scavenging effects on DPPHradicals
One hundred microliters of various concentration of the tea
polysaccharide were mixed with 2900 L of DPPH solution
(120 M) in ethanol and incubated in darkness at 37 C for 30 min.
The absorbance was recorded at 517 nm. Percentage (%) inhibition
of tea polysaccharide on free radical production by DPPH was cal-
culated (Liu and others 2007). Ascorbic acid was used as positive
control, and all tests were carried out in triplicate.
Inhibitory effects on nonenzymatic lipid
peroxidation induced by Fe
2+
/ascorbate
The inhibition of lipid peroxidation was assayed by the method
of Anup and others (2006) with slight modifications. Kunming mice
weighing 20 2 g (purchased from Beijing Weitonglihua Co., Bei-
jing, China) were housed under conventional conditions and were
allowed free access to food and water. All experiments were carried
out according to the guidelines for the care and use of experimental
animals and approved by state veterinary administration.
The mice were anesthetized using diethyl ether and the ab-
domen was opened and the liver was quickly removed. The livers
were then cut into small pieces and homogenized in phosphate
buffer (50 mM, pH 7.4) with a homogenizer to give a 10% (w/v)
liver homogenate. The liver homogenate was further centrifuged
at 3000 g for 10 min. The supernatant of the liver homogenate
was collected and the amount of protein was determined by the
method of Bradford (1976). The extent of lipid peroxidation was
evaluated by measuring the product of thiobarbituric acid-reactive
substances (TBARS) in the rat liver homogenate. The reaction mix-
ture was composed of 0.5 mL of tissue homogenate, 0.9 mL of phos-
phate buffer (50 mM, pH7.4), 0.25 mL of FeSO
4
(0.01 mM), 0.25 mL
of ascorbic acid (0.1 mM), and 0.1 mL of different concentration of
the extract and the standard sample. The reaction mixture was in-
cubatedat 37